Félix Sauvage

Efficient and safe intracellular delivery of macromolecules, like nucleic acids, remains a major challenge in ex vivo cell engineering. While photoporation is a highly attractive method for cytosolic delivery of (macro)molecules, photosensitizers remaining in the photoporated cells may pose safety concerns, especially when the cells are engineered for therapeutic use. In this study we introduce the concept of “detachable photosensitizers” using gold nanoparticles (AuNPs) as photosensitizer. We demonstrate that phenylboronic acid (PBA)‐functionalized AuNPs can be anchored to membranes of HeLa cells and Jurkat cells, thereby enabling the efficient intracellular delivery of dextrans and mRNA upon pulsed laser irradiation (photoporation). Subsequently, the PBA‐AuNPs can be detached from the cells “on‐demand” by adding ATP to the cell medium, as cis‐ diol groups in ATP displace the boronate ester bonds between the PBA‐AuNPs and cell‐surface. ICP‐MS analysis demonstrated efficient detachment, with the average cell‐associated gold mass per viable cell falling below the theoretical mass of a single intact 80 nm AuNP. This “attach‐and‐detach” strategy offers a robust and effective approach to addressing safety concerns associated with the use of plasmonic nanomaterials as sensitizers for photoporation, and holds significant potential for the safe manufacturing of engineered cells in the context of cell therapy.

Diseases of the ocular surface, such as dry eye disease (DED), can significantly impair vision and reduce the quality of life. Current treatments rely primarily on conventional approaches, including artificial tears, antibiotics, and anti‐inflammatory drugs. However, advances in nucleic acid (NA) therapeutics have opened new avenues for treating ocular surface disorders, with growing interest in small interfering RNA (siRNA) and messenger RNA (mRNA) as potential drugs. A key challenge remains the efficient delivery of these macromolecules into cells of the ocular surface, where both extracellular and intracellular barriers hinder their cellular uptake and activity. To address this limitation, we explore the use of photoporation, a laser‐based physical transfection technique, to enhance mRNA delivery into ocular surface cells. Photoporation employs pulsed‐laser irradiation of photosensitizers to generate vapor nanobubbles (VNBs), whose rapid expansion and implosion produce mechanical forces that transiently permeabilize cell membranes, thereby promoting intracellular delivery of co‐administered nucleic acids. Using polydopamine nanoparticles (PD NPs) as biocompatible photosensitizers for photoporation, we demonstrate mRNA transfection in the corneal epithelium with both an experimental and a clinically approved pulsed‐laser system. Our results highlight the potential of laser‐based approaches for noninvasive engineering of the ocular surface.

Hsp90 (heat shock protein 90) est une protéine chaperonne ubiquitaire et conservée impliquée dans le repliement et la réparation de protéines dites « clientes ». Parmi ces protéines, de nombreuses sont impliquées dans des phénomènes oncogéniques, faisant de hsp90 une cible d’intérêt dans le traitement du cancer. Hsp90 est constituée de trois domaines, un domaine N-terminal site de l’hydrolyse de l’ATP, nécessaire à sa fonction ; un domaine intermédiaire où vient se fixer la protéine cliente et un domaine C-terminal impliqué dans la dimérisation, étape indispensable pour le repliement de la protéine cliente. De nombreux inhibiteurs ont été synthétisés en ciblant ces différents domaines. Les inhibiteurs N-terminaux sont efficaces, à l’instar de la Geldanamycine en termes d’activité anti-tumorale mais des effets secondaires ainsi que des résistances au traitement ont limité leur utilisation en pratique clinique. En effet, l’inhibition N-terminale induit une réponse au stress caractérisée par une augmentation de hsp90 et de ses co-chaperonnes, souvent associée à une résistance au traitement et un pronostic défavorable. La novobiocine, un antibiotique coumarinique, est capable d’inhiber le domaine C-terminal de hsp90, sans induire de réponse au stress. Ainsi de nombreux dérivés de cette molécule ont été synthétisés, parmi lesquels on trouve le 6BrCaQ. Cette molécule induit l’apoptose et le blocage dans le cycle cellulaire sur plusieurs lignées cellulaires (dont MCF-7 et MDA-MB-231) et provoque la dégradation de plusieurs protéines clientes impliquées dans le développement tumoral mais sa faible solubilité limite son administration in vivo.Dans cette thèse, une forme liposomale du 6BrCaQ a été développée et étudiée sur des lignées cellulaires de cancer de prostate, de sein et de leucémie aigüe myéloïde in vitro et in vivo sur un modèle orthotopique de cancer du sein (MDA-MB-231 luc-GFP). Le 6BrCaQ liposomal est capable d’ induire de l’apoptose, de bloquer le cycle cellulaire sur différentes lignées cellulaires (PC-3, MDA-MB-231 et MOLM-13) mais également de ralentir la migration cellulaire sur PC-3 (test de comblement de blessure). De plus, le 6BrCaQ liposomal entre en synergie avec la doxorubicine (cellules PC-3) et la daunorubicine (cellules MOLM-13). Au niveau moléculaire, les liposomes de 6BrCaQ modifient l’expression protéique de Hsp90 sans modifier celle d’Hsp70 sur PC-3 alors que les gènes codant pour les Hsp70 semblent être légèrement induits dans MDA-MB-231. Les résultats in vivo ont montré un ralentissement de la croissance tumorale sur un modèle de cancer du sein orthotopique (MDA-MB-231-luc2-GFP) dès 13 jours de traitement pour une dose de 1 mg/kg injectée une fois par semaine. Des analyses histologiques ont révélé une augmentation de la proportion des zones nécrotiques dans le groupe traité par rapport au contrôle et une diminution significative de la prolifération cellulaire (marquage au KI67) intra-tumorale.Par ailleurs, Hsp90 possède également des isoformes et des analogues localisés dans des organites intracellulaires, parmi lesquelles, TRAP-1, localisée au niveau de la mitochondrie, impliquée dans la rgulation du métabolisme mitochondrial et qui pourrait jouer un rôle dans la progression tumorale et les métastases. Le déqualinium (DQ) est capable de cibler la mitochondrie. Dans une seconde partie du travail, des liposomes encapsulant le DQ ont été formulés dans le but de vectoriser le 6BrCaQ vers la mitochondrie. Toutefois, face à la difficulté d’encapsuler le DQ dans des liposomes, une étude de physico-chimie sur l’interaction DQ/liposomes a été mise en place pour comprendre comment le DQ agit sur les bicouches phospholipidiques. Cette étude a révélé que, malgré une capacité de ciblage mitochondrial des liposomes, le DQ était difficile à encapsuler dans les milieux salins et n’était pas inerte sur les bicouches lipidiques ce qui limite son utilisation pour la formulation de liposomes ciblant la mitochondrie.

Having access to high‐quality cell populations is essential for biomedical research, diagnosis, prognosis, and therapeutic applications. Traditional enzymatic methods, such as trypsinization, remain widely used to detach cells from supporting material surfaces, though they often compromise cell surface integrity, viability and functionality. To address these limitations, diverse strategies have emerged that enable a much gentler release of cells from material surfaces. The use of external physical stimuli (like shear stresses, light, temperature, magnetic fields), chemical stimuli (based on changes in pH or ligand‐exchange based detachment) and biological stimuli (like aptamers and nucleic acid sequences) have been explored for the purpose of non‐invasive cell detachment. This review discusses the principles, mechanisms, advantages, and limitations of these most diverse cell detachment technologies. By providing this comprehensive perspective, we hope to guide researchers in selecting the most suitable techniques for their specific needs and to inspire further innovation in this vital biomedical area.

Photothermal nanomaterials enable precise tumor ablation but face limitations in biodistribution, tissue penetration, toxicity, and biodegradability. Here, we present a unique concept for nanoparticle-free photothermal therapy based on the lysosomal entrapment of cationic amphiphilic small molecular dyes for spatially controlled vapor bubble (VB)-mediated tumor cell ablation. This strategy, which exploits a universal biological and physical effect, employs intracellular pH gradients for extensive local dye enrichment in acidified organelles, transforming them into transient endogenous nanosized photothermal reactors for subsequent light activation. Using sunitinib, a clinically approved lysosomotropic anticancer drug, and the commercially available dye LysoTracker™ Deep Red, lacking intrinsic anticancer activity, we demonstrate pulsed laser-induced VB formation specifically from dye-loaded lysosomes, leading to selective photomechanical disruption of various cancer cell models across 2D cultures, 3D spheroids, patient-derived neuroblastoma tumoroids and tumor fragments from an ovarian carcinoma patient. This approach allows precise, low-fluence and wavelength-tunable cancer tissue ablation without the need for synthetic photoresponsive nanoparticles.

Visual impairments affect over 2.2 billion people worldwide, yet delivering drugs to the posterior segment of the eye, including the retina, remains a major challenge. Intravitreal injection, the standard administration route, often results in suboptimal drug diffusion through the vitreous, limiting drug access to the retina. While various strategies have been explored to enhance the mobility of drug molecules and nanomedicines (drugs encapsulated in nanoparticles) in the vitreous, no method has demonstrated guided transport . Here, we investigate photothermal transport of nanoparticles in the vitreous using a pulsed laser and indocyanine green, both clinically approved modalities. We show that photothermal transport guides nanoparticles from one location in the vitreous toward the laser‐illuminated area, away from the injection spot. Multiple‐particle tracking and numerical simulations reveal that this motion is predominantly driven by thermal convection, with thermophoresis contributing to a lesser extent. We identified parameters for optimization, including dye concentration, particle size, distance from the laser focus, and laser fluence. These findings establish a novel and clinically relevant paradigm for light‐guided drug delivery in the eye. To our knowledge, this is the first demonstration of guided light‐controlled particle transport in the vitreous using ocular dyes and pulsed lasers routinely applied in ophthalmology.

Collagen aggregation in the vitreous is a major cause of vision impairment. Current treatments such as vitrectomy or YAG laser vitreolysis remain limited by invasiveness and safety concerns. In previous work, we introduced a novel approach combining indocyanine green (ICG) with nanosecond laser pulses to achieve photodisruption of collagen aggregates via vapor nanobubbles (VNBs), while using a significantly lower total light dose than that applied in clinical laser vitreolysis. However, despite its clinical approval, free ICG poses a risk of retinal toxicity. In this work, we report the development of ICG nanoaggregates (ICG AGG NPs) stabilized with a minimal amount of a hyaluronic acid (HA)-lipid (DOPE) conjugate designed to limit retinal penetration of ICG while preserving efficient VNB generation and collagen aggregate disruption. We demonstrate that supramolecular aggregation is a key requirement for efficient VNB generation, whereas encapsulation of ICG in conventional liposomes impairs this process. Using a newly established in vitro model for quantifying collagen disruption, we show that ICG AGG NPs significantly enhance photodisruption compared to free ICG. Furthermore, cell toxicity assays on retinal pigment epithelium (RPE) and Müller cells indicate that ICG AGG NPs maintain an acceptable safety profile at therapeutic concentrations. These findings represent the first successful demonstration of dye-loaded nanoparticles enabling efficient VNB-mediated photodisruption of vitreous opacities and highlight the promise of ICG AGG NPs as a safer and more effective alternative to free ICG for floater treatment.

Visual impairments affect over 2.2 billion people worldwide, yet delivering drugs to the eye’s posterior segment, including the retina, remains a major challenge. Intravitreal injection, the standard administration route to the posterior segment of the eye, often results in suboptimal drug diffusion through the vitreous, preventing drugs from reaching the retina. While various strategies have been explored to enhance the mobility of drug molecules and nanomedicines (drugs encapsulated in nanoparticles) in the vitreous, no method has demonstrated ‘guided transport’ of drugs and particles through the vitreous. In this study, we explore photothermal transport of nanoparticles in the vitreous using a pulsed-laser and indocyanine green added to the vitreous, both being clinically approved modalities. We found that photothermal transport allows to guide nanoparticles from one place in the vitreous towards the laser illuminated area in the vitreous, at a distance of the injection spot of the nanoparticles. Using multiple-particle tracking and numerical simulations, we found that both thermal convection and thermophoresis contribute to photothermal transport of nanoparticles in the vitreous. We identified parameters for optimizing this effect, including dye concentration, particle size, distance from the laser focus, and laser fluence. Our findings establish a novel and clinically relevant paradigm for light-guided drug delivery in the eye. This study represents, to our knowledge, the first demonstration of guided light-controlled particle transport in the vitreous using ocular dyes and pulsed-lasers which are routinely used in ophthalmology.